Abstract 12718: Hydrogen Peroxide Differentially Modulates Cardiac Myocyte Nitric Oxide Synthesis
Nitric oxide (NO) and hydrogen peroxide (H2O2) are synthesized in cardiac myocytes and play key roles in cardiovascular signaling. Cardiac myocytes contain both endothelial (eNOS) and neuronal (nNOS) NO synthases, but the differential roles of these NOS isoforms and the interplay of reactive oxygen species and reactive nitrogen species in cardiac signaling pathways are incompletely understood. Using the new NO chemical sensor CUFL2 to study NO synthesis in adult cardiac myocytes from wild-type, eNOSnull, and nNOSnull mice, we discovered that physiological concentrations of H2O2 (25 μM) activate eNOS but not nNOS. H2O2-stimulated eNOS activation depends on phosphorylation of the AMP-activated protein kinase, and leads to phosphorylation of eNOS at residues S633 and S1177, associated with striking increases in NO synthesis. We next explored the receptor-modulated control of endogenous H2O2synthesis in these cells using the novel H2O2 biosensor HyPer2. We cloned HyPer2 into lentivirus and introduced this construct into adult mice via tail vein injection, which yielded robust biosensor expression in the heart and in other tissues. Two weeks after injecting mice with the HyPer2 lentivirus, we isolated cardiac myocytes and used quantitative fluorescence microscopy to analyze responses to angiotensin-II (500 nM) or to the beta adrenergic receptor agonist isoproterenol (100 nM). Using live cell imaging of HyPer2 fluorescence, we found that angiotensin-II but not isoproterenol activated H2O2 synthesis in these cells. We incubated cardiac myocytes with PEG-catalase to degrade intracellular H2O2, and found that angiotensin-II- but not isoproterenol-promoted NO synthesis (detected with the CUFL2 NO sensor) was blocked by PEG-catalase. These studies establish differential roles for H2O2in receptor-dependent NOS activation in the heart and identify new points for modulation of NO signaling responses by oxidant stress.
- © 2011 by American Heart Association, Inc.